Present situation of artificial cultivation of sexual Cordyceps sinensis

Over the past 20 years, more than 60 scientific research units and thousands of people have carried out a lot of research, experiments and explorations on artificial cultivation of Cordyceps sinensis. Yunnan, Sichuan and other places have achieved successful experience in cultivating sexual types in small areas at high altitude, but due to many factors such as long cycle, few promotion objects, large investment and lower income than natural collection at high altitude, it has not been popularized in large areas. In the middle and low altitude areas of Yunnan, Sichuan, Zhejiang, Gansu, Qinghai, Beijing, Fujian, Guangdong, Guizhou, Guangxi and other provinces (cities, districts), the research on artificial cultivation of sexual Cordyceps sinensis has also been carried out for many years, although many of them have achieved very gratifying results in some aspects, such as the successful breeding of moth insects in Kunming, Chengdu and Chongqing in Yunnan and Sichuan provinces. In Zhejiang and other places, bats and moths have successfully propagated indoors; However, it can't be called successful culture because it doesn't solve the difficulties of inoculation of entomogenous bacteria and batch and repeated growth of progeny. It is still far from the goal of artificially cultivating sex Cordyceps sinensis in small area, small batch and many times at low altitude. Although several companies claim that they have successfully cultivated sex Cordyceps sinensis in batches at low and medium altitudes, the scientific credibility is extremely low in fact. Because:

(1) At present, no one has been able to isolate and cultivate authentic Cordyceps fungi stably for a long time (that is, a large number of daughter bases have been repeatedly grown on the culture medium). Substrates have been cultivated in Qinghai, Yunnan, Sichuan, Beijing and other places, but their ascospores are few or none, and their growth is unstable, which does not mean that they have mastered the sexual artificial cultivation technology at middle and low altitudes.

(2) The artificial asexual culture technology of Cordyceps sinensis is relatively easy to master. Inoculation of hepialus larvae indoors can lead to infection and enter into a stiff stage, but it is rare to complete the sexual form and grow the stroma and ascospores.

(3) Apart from environmental factors such as altitude, climate, soil and air pressure, are there multi-bacterial infections among bacteria? If it is a compound infection, which bacteria are playing a key role? When did it work with Cordyceps sinensis? Wait, the question is not clear.

Therefore, it needs a lot of research and experimental work to batch and repeatedly cultivate Cordyceps sinensis in middle and low altitude areas. Even if large-scale artificial cultivation of Cordyceps sinensis is carried out in high altitude areas, many technical experiments are still needed. Therefore, it is still very arduous and far away to cultivate sex Cordyceps sinensis artificially and realize large-scale or industrial production.

(2) Current situation of artificial culture of asexual Cordyceps sinensis.

Because it is too difficult to cultivate sexual Cordyceps sinensis artificially, in recent years, most units and researchers who have cultivated Cordyceps sinensis artificially have turned to asexual research and application. Because the asexual culture technology of Cordyceps sinensis is easy to master, and most of the fungi isolated from Cordyceps sinensis collected in nature have similar physical and chemical properties and pharmacological effects to the sexual components of Cordyceps sinensis, all localities have identified the self-isolated fungi as the asexual stage of Cordyceps sinensis, and have set up factories to produce bacterial powder for development and application. At present, there are 9 genera and 3 1 species of Cordyceps sinensis isolated from the body of Cordyceps sinensis in nature, including 16 species found in more than 40 provinces (cities) such as Beijing, Jilin, Shanxi, Shanghai, Qinghai, Zhejiang, Fujian, Jiangxi, Guangdong, Yunnan and Guizhou. Submerged large-scale fermentation culture and solid culture of mycelium and conidia are substitutes for sex Cordyceps sinensis, and some manufacturers have entered large-scale industrial production, such as more than 20 kinds of products such as Lexus capsule, Jinshuibao capsule, Ningxinbao capsule, Xinganbao, Cordyceps sinensis chicken essence, Cordyceps sinensis capsule, Cordyceps sinensis wine, Cordyceps sinensis ginseng tonic wine, etc., with an annual output value of10 million yuan to hundreds of millions. Moreover, due to the short artificial propagation cycle and high yield, mycelium and meristem robe have great development and application potential as sexual substitutes of Cordyceps sinensis in the future.

In order to obtain pure strains of grass in winter and early summer, it is necessary to separate, purify, rejuvenate and preserve them artificially.

1. separation technology and conditions of cordyceps sinensis

(1) Material requirements. Obtaining pure strains requires strict materials; Generally speaking, if the tissue block separation method of Cordyceps sinensis is adopted, the best separation material is the material collected from the end of 65438+1October every year to165438+1October, when the soil in the alpine meadow on the Qinghai-Tibet Plateau has just begun to freeze. During this period, Cordyceps sinensis just infected moth larvae, and entered the stage of zombie for a short time. The tender bud just grew a worm head of 0.2-0.5cm, and there were few other miscellaneous bacteria in the worm. If the samples are taken in May and June of the following year, there are many kinds of miscellaneous bacteria living or saprophytic in the stiff worm and its sub-seat, so it is difficult to obtain pure strains. If the ascospores are used for isolation and culture, it is best to collect the isolation materials in the middle and late July of each year when the ascospores are just partially mature.

(2) Operation method. There are three steps.

A. separation of stiff worms (i.e. sclerotia): before separation, the surface of the main body is washed with water, and then washed with sterile water for 2-3 times; Disinfect the surface of the separated material with 0.65min438+0%-0.2% mercuric chloride solution for about 3-5min; Then wash with sterile water. Select the front part bounded by the chest and feet. Cut off the epidermis with a surgical knife, avoid the digestive tract, cut the hyphae in the blood cavity into sesame seeds, and press them into the plate culture medium, each dish contains 1-2 capsules. Cultured at 15- 19℃. When the colony grows to 0.2-0.5cm, select a small amount of mycelium and purify it repeatedly on the plate medium for 2-4 times. After confirming that there are no other miscellaneous bacteria, transfer to a test tube for preservation and culture.

B, separating the bud: cutting the washed bud from the top of the stiff worm, putting it into 0. 1% mercuric chloride solution for disinfection for 2-3min, washing it with sterile water, cutting off the middle tissue block and pressing it into the culture medium; The culture conditions are the same as a.

C. Ascomycete spore separation: covering the mature substrate of Ascomycete spores with a transparent paper bag, and allowing the Ascomycete spores to bounce and stick to the paper bag. Then, the paper bag containing ascomycete spores was immersed in 25% glucose solution, washed off the spores, and cultured at 15- 19℃, and examined microscopically every day. When the robe germinates, a single spore is sucked onto the plate with a micropipette for culture. The culture conditions are the same as those in A, or the whole mature Cordyceps sinensis can be brought indoors, and the stiff insect or even its daughter seat can be wrapped with cotton paper, leaving only the pregnant part, and the glass slide can be placed horizontally in the sterile room to keep the humidity of the main body at any time; Microscopic examination every day, when ascospores bounce on the glass slide, they are sucked into the culture medium of Petri dish with a micropipette for culture; The culture conditions are the same as a.

(3) Selection of culture medium.

A. Potato glucose agar medium (PDA) is a universal medium for all Cordyceps fungi, which can be used in the initial stage of isolation and culture, but the strain growth is not very vigorous and it is easy to age and degenerate.

B enrichment medium (1): multifunctional protein umbilical cord 10g, glucose 50g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, live silkworm chrysalis 30g, auxin 0.5μg, agar 20g, water 1000mL, pH: 5.0.

Enrichment medium (II): 40g of preserved protein, 40g of glucose, peeled fresh potato 100g, dipotassium hydrogen phosphate 1g, 0.5g of magnesium sulfate, beef paste 10g, 0.5μg of auxin, 30g of bat moth live larvae (powder), 20g of agar and alpine meadow soil leachate/kl.

On the intensive medium, the colonies grew more vigorously and rapidly than on PDA medium. Jiafu Ⅱ is superior to Ⅰ.

2. Growth characteristics In the separation and culture of Cordyceps sinensis, it is necessary to master the utilization of temperature, pH value, illumination, carbon and nitrogen sources and inorganic salts.

(1) Effects of different temperatures on mycelium growth. Cordyceps likes low temperature. It can grow slowly at 0-4℃; The growth rate is accelerated at 5-8℃; 10- 19℃ is the suitable growth and development range of this strain; The optimum growth temperature is 15- 19℃. When the temperature exceeds 20℃, the mycelium grows rapidly, the colony changes from white to grayish black or brownish yellow, and begins to mutate, so it is not suitable for high temperature culture.

(2) Requirements for pH value. Cordyceps sinensis is an acid fungus, and its optimum pH value is 5.0-6.0. When the pH value is below 4.5 and above 6, it will increase slowly or not with the increase or decrease.

(3) the influence of light. The strain adapts to weak light and short light in the early stage of ascospore germination and mycelium growth, and adapts to strong light in the later stage. In artificial culture, hyphae, conidia and substrate have obvious phototaxis, which grow densely in the sunny side and sparsely in the backlight side. All kinds of bacteria cultured in the all-black environment are delicate, slender and sparse.

(4) Utilization of carbon source. Cordyceps sinensis can use a variety of carbon sources, but when glucose and maltose are used together, it grows fastest, and when glucose is used alone, it can grow well, followed by starch such as potato and sucrose.

Cordyceps sinensis is a fungus that grows from a parasitic host to a saprophytic stage, so it can make good use of various organic nitrogen sources. And live worms are the best; The mixed use of peptone and yeast extract is also very ideal for growth, followed by their own use, followed by beef sauce and so on. However, the utilization of inorganic nitrogen such as ammonium sulfate and potassium nitrate is poor. There is a certain demand for ash nutrients, which grow vigorously in the medium containing trace amounts of magnesium sulfate and dipotassium hydrogen phosphate, followed by potassium dihydrogen phosphate, and other inorganic salts such as sodium, calcium, iron and copper can also be used.

3. After separation and purification, Cordyceps sinensis can be used as various experiments and expanded production applications.

(1) There are usually three methods to increase its yield: solid static culture, shake flask culture and large tank aeration fermentation culture.

A. Static culture: mainly used for solid culture, such as tube slant culture, triangular flask culture, rice culture, etc. In static culture, bacteria can grow normally as long as the temperature and light are well controlled. The conidia on the inclined plane can be stored in the refrigerator at 1-2℃ for 8- 14 months after maturity, and can also be directly used as seeds for production.

B. shaking culture: both liquid culture and small-scale propagation culture can be used. Shake the medium and subtract agar from the solid medium. It is best to choose a constant temperature oscillating culture machine, put the liquid culture medium into a triangular bottle and inoculate the test-tube solid strain for culture. After continuous oscillation, the components in the liquid medium are mixed evenly without precipitation, and at the same time, gas-liquid contact and exchange are promoted, so that oxygen enters the liquid medium, which is beneficial to mycelium growth and conidia formation.

C. aeration fermentation culture in large tanks: When producing mycelia, conidia and other bacterial powders on a large scale, aeration fermentation culture in large tanks must be used. In this method, aeration is carried out through suction or vacuum pump decompression, and bacteria are removed through a filter, and then they are sent to a liquid culture medium in a tank for the growth and development of Cordyceps sinensis.

(2) Fermentation process. Separation and purification of natural Cordyceps sinensis → slant seeds in test tubes → primary seeds of oscillating liquid → secondary seeds → small-scale seed fermentor → production fermentor → concentration → powder spraying and drying → finished bacterial powder.

The fermentation temperature of Cordyceps sinensis in the tank is 20-25℃, the tank pressure is 392.3-686.5kPa(0.4-0.7kg/cmz), and the ventilation rate is 0.5- 1.ovvm *. The liquid culture medium injected into the fermentor should be 65%-75% of the tank capacity. The inoculation amount is 10%, the stirring speed is 180 rpm, and the culture time is 72-96h, so that it can be concentrated and dusted.

(3) Multi-purpose fermentation medium. Fresh potato (peeled) 8%, sucrose 2%, corn starch 0.5%, silkworm chrysalis powder 1%, peptone 0.4%, ammonium sulfate 0.2%, and pH value 5.5-6.0.

(4) the standard of fermentation culture products. Conidia are almost completely separated female spore stalks. When the number of spore stalks did not increase obviously, the number of conidia per ml was 654.38 0.8 billion ~ 2.5 billion. Residual sugar is less than1%; When the amino nitrogen is lower than 0.2mg/mL, it can become the finished bacterial liquid.

4. Inoculation points of Cordyceps sinensis: Inoculating host insects with sexual or asexual Cordyceps sinensis spores is a key measure to artificially cultivate sexual Cordyceps sinensis and rejuvenate asexual strains.

(1) tieback period. The key to the reconnection of Cordyceps sinensis is to choose the weak antibacterial period of the host insect, one is the molting period; The second is the period when the larval feeding activity is intense and the friction damage rate is high.

(2) tieback method. There are two kinds.

A. Spraying method: When the hepialus moth larvae molt at the age of 4-6 and above 65,438+0/3, the larvae are concentrated and sprayed with 5%-65,438+00% glucose solution on the insect body or food with ascospores or conidia. After inoculation, the bacterial liquid is slightly dried on the insect body for about 30 minutes, and the larvae are put back into the soil to live naturally. This method has a high infection rate, but the disadvantage is that the larvae are not easy to be taken out of the soil and concentrated, and the larvae have the habit of killing each other when concentrated. After being bitten, bugs are easily infected by miscellaneous bacteria and die. When the mycelium of Cordyceps sinensis does not fill the body cavity, the injured worm will rot and cannot form a rigid worm (sclerotium).

B. Natural contact method: feeding hepialus moth larvae in a large area of semi-natural soil, and regularly taking small samples to observe the growth of the larvae. When most of the larvae are suitable for infection, fine soil is planted and inoculated with feed plant soaking solution, which is evenly spread on the surface of the raised insects, and then water is sprayed to make bacteria penetrate into the soil, so that the larvae are exposed to infection during feeding activities. The infection rate of this method is not too high, but once infected, Cordyceps sinensis grows well, and uninfected hepialus larvae can also grow and reproduce normally.

(Excerpted from Yang's Utilization and Industrialization of Insect Resources in China) Excerpted from Utilization and Industrialization of Insect Resources in China.

Cultivation techniques of early summer grass in winter

(a) an overview of Cordyceps sinensis alias, Cordyceps sinensis, Cordyceps sinensis. It is cordyceps sinensis (Berk. )Sacc。 A plant belonging to the genus Cordyceps of ergot family, and the dried insect corpse of its strange owner. Gan, Wen Nourishing lung and tonifying kidney is mainly used to treat tuberculosis, hemoptysis, asthenic asthma, night sweats, nocturnal emission, impotence and soreness of waist and knees. Mostly in mountains, grasslands, river valleys and grasslands. Distributed in Gansu, Qinghai, Sichuan, Yunnan, Tibet and other provinces.

(2) Plant characteristics Cordyceps sinensis is a combination of fungi belonging to Ascomycetes, Carnivora, Ergonomidae and Cordyceps, which are parasitic on Lepidoptera moth larvae. The whole shape is composed of insect body and fruiting body, and the appearance is yellow-brown or yellow-brown. Much like a silkworm, the worm is slender and cylindrical, 3 ~ 5 cm long and 0.3 ~ 1 cm thick, with 20 ~ 30 obvious links, 8 pairs in the abdomen, especially 4 pairs in the middle. The fruiting body refers to the part that extends outside the host insect. After repeated knotting and differentiation of hyphae, the reproductive organs can be recognized by the naked eye. Crispy texture, yellow-white cross section, reddish-brown head and pods. Pods are mostly solitary, slender as a stick, 4 ~ 10 cm long, swollen at the top, brown in surface, slightly tough in texture, yellowish white, and fibrous in cross section. There are scattered or dense ascomycetes in the head of the pod, and countless ascomycetes are formed in the ascomycetes, which are ascospores that can reproduce sexually.

(3) Growth characteristics The distribution of Cordyceps sinensis is closely related to topography, landforms and altitude. Its main moths like to grow on wet, low temperature, sunny and well-drained topography and landforms.

(4) Cultivation techniques

1, culture conditions Artificial culture of Cordyceps sinensis is mainly to prepare strains and insects.

The cultivation of (1) strain Cordyceps sinensis should first have excellent pure strains, and first have early maturity and high yield. The main purpose is to shorten the production cycle and reduce the cost. Second, it should be contagious, requiring the strain to have strong vitality and a survival rate of over 95%, and it can quickly infect insects and get sick and die as soon as possible. Third, it has a wide range of application, especially for miscellaneous bacterial infections such as environmental temperature changes.

(2) Insects mainly use the larvae of Babesia moths as parasites of Cordyceps sinensis. Larvae must be alive, large and obese, and the number depends on their own breeding. Generally, every square meter needs larva 1 kg, mother seed 1 grain, 50 kg of fine sand, and can grow 0.5 kg of fresh cordyceps and 0.05 g of dried products, with a value of 100 yuan.

(3) Cordyceps sinensis can be cultivated in an artificial environment regardless of altitude, and the key is temperature. Cordyceps sinensis is a medium-low temperature fungus. The optimum temperature for mycelium growth and propagation is 5 ~ 32℃, the optimum temperature is 12 ~ 18℃, and the optimum temperature for sclerotia and matrix formation is 10 ~ 25℃.

(4) Natural temperature can be used in the cultivation season, and it can be cultivated twice a year, from March to May in spring and from 9- 165438+ 10 in autumn. If the temperature is controlled indoors, it can be cultivated all year round and the growing period can be shortened.

2. Cultivation methods Cordyceps sinensis cultivation methods include indoor and outdoor bottle cultivation, box cultivation, bed cultivation, open field cultivation and so on. And you can choose any one according to your own conditions. No matter what kind of cultivation method, bacteria and insects should be cultivated before cultivation, so that insects can be infected with this virus-carrying bacterial solution before burial, and they can't climb everywhere after burial, which is conducive to early death, rapid emergence and orderly growth.

The cultivation method of bacteria and insects is to spray the prepared liquid strain on the larvae with a sprayer twice a day until it is wet. After 3 days, the larvae soaked in the bacterial liquid appear sluggish and coma, which can be cultured.

(1) Bottle cultivation is suitable for family cultivation. After the ordinary canned bottle is cleaned, first put a layer of fine sand with soil water content of 60% in the bottle, and then put the larvae infected with bacterial liquid on it. It is advisable to put two larvae in each bottle. It is required that the two larvae do not approach each other, with their ventral faces down and their surfaces stretched, covered with fine sand for 3 cm, and their surfaces slightly flat. In order to keep moist, it should be sealed with plastic film and managed at suitable indoor and outdoor temperatures to avoid direct sunlight.

(2) Box cultivation is also suitable for family cultivation, and both large and small wooden boxes and plastic pots can be cultivated. There should be plastic film at the bottom and around the wooden case to prevent water loss. Spread the fine sand with a thickness of 5-7 cm first, then evenly put the bacteria and worms with a spacing of 2-3 cm, and then cover with a sauce of 3-5 cm. The surface should be moisturized with plastic film. To save space, you can also overlap the wooden boxes.

(3) Bed-planting and bedstead cultivation is a development mode of large-scale production, which is generally suitable for indoor planting, and can make full use of indoor space for planting and save space. Bed frame width 100 cm, according to the design of your own room, made of bamboo and wood. The four sides of each floor are 12 cm high, which is used for retaining soil. When cultivating, first spread a layer of plastic film, then pour 5-7 cm of fine sand to flatten it, then put in bacteria and insects, plant them in boxes every 3-5 cm, and then cover them with plastic film.

(4) Open cultivation refers to outdoor cultivation. The key to outdoor cultivation is to choose a good site. First, it is necessary to avoid direct sunlight and rain erosion, and achieve an environment that can shade, drain, prevent drought and prevent people and animals from trampling. There are two cultivation methods: flat type or border type. Flat land cultivation is to remove the topsoil of ordinary flat land, covered land and wasteland with a width of 100cm and an unlimited length, then fill in sand with a thickness of 5 cm, put in bacteria and insects according to the above method, cover it with fine sand of 5-7 cm, and cover it with plastic film. There should be a drainage ditch around, and trees or shade sheds should be built on it for shade.

(5) Border tillage can avoid the problems of sunshine and high temperature and is suitable for rural farming. Border width 100 cm, depth 50 cm, unlimited length. The surrounding area can also be drained. During cultivation, fine sand with a thickness of 5 cm is laid on the bottom of the border, then bacteria and insects are put in according to the above method, then 5 cm is covered on the fine sand, and finally a plastic film is covered. Bamboo arches are used beside the frame to cover the grass curtain for shading and cooling.

3. Management technology The management technology of Cordyceps sinensis after cultivation is very simple, mainly in terms of temperature, humidity, light and air.

(1) temperature Cordyceps sinensis requires a wide temperature, which is generally low first and then high, but if it is too low, it would rather grow slowly, and if it is too high, it will not be affected. The mycelium grows at 12- 18℃, but the survival rate is high at low temperature. Generally, -40℃ will not freeze to death, but it will freeze to death when it is higher than 40℃, and 20 ~ 25℃ is beneficial to the growth of progeny in the later stage.

(2) Humidity and humidity management are the key to the growth and development of Cordyceps sinensis. The nutrition and humidity in insects can basically meet their growth needs. It does not need external nutrition and humidity, but only external objects to keep the insect body at a temperature that is not easy to dry. Therefore, it is advisable to keep the sand moist at any time, and the water content should reach 60%. If it is dry, you can spray a small amount of water to keep it moist.

(3) Illuminated Cordyceps sinensis cultivation does not need strong light, and it is better to avoid light. It is better to use astigmatism in the later stage of development, but it can't let the sun shine directly. Especially for outdoor cultivation, it is necessary to cover the shade with tree-lined, artificial shade shed and grass curtain.

(4) A lot of air is not needed in the growth stage of airborne Cordyceps sinensis mycelium, especially when the pedestal is about to leave the ground, the plastic film should be removed immediately and air should be supplemented to facilitate the growth of the pedestal, and the relative humidity of air should be kept at about 75% ~ 95%, and it will mature after excavation 10 ~ 20 days.

(5) Harvesting and processing Under natural conditions, the growth period of Cordyceps sinensis is generally 9 months, and the maturity standard of variety traits mainly determines the growth rate. It can be harvested when the substrate is 3 ~ 5 cm high and the top of the substrate develops into a "hairtip" of ascocarp.

Harvest method: gently dig the root beer with bamboo poles and wooden poles, pick out Cordyceps sinensis and put it in the basket. Be careful not to damage the insect body and the auxiliary seat, let alone scratch the insect body or the auxiliary seat. After harvesting, wash the sediment with water and put it in the sun or dry it in time.

Processing method: The packaged Cordyceps sinensis is processed with loose Cordyceps sinensis as raw material. That is to say, the loose cordyceps sinensis should be straightened after the resurgence, tied into small bundles every 7 ~ 10, and baked with low fire until it is completely dry. The tail of 48 small bundles should be put into iron grids and packed in three layers, with more than 6 pieces per layer/kloc-0. After extrusion, it should be fumigated and dried, and the trademark should be fastened with red silk rope. According to the specifications, each letter of Cordyceps sinensis should be kept at about 0.25g, packed in wooden cases, covered with a layer of moisture-proof paper, tied with iron hoops and stored in a ventilated and dry place.

Article source: Tang Hanzhong Medicine Business Network

Second, artificial cultivation of Cordyceps sinensis:

Artificial cultivation should solve at least four problems.

One is stress. Most of the strains are Cordyceps sinensis from nature, which are obtained by routine separation and culture. According to the research, during the sexual morphogenesis of Cordyceps sinensis, a certain temperature is needed to meet the water needed for complex physiological changes in the body, and there is a correlation between temperature and humidity. Under a certain humidity, slow or constant temperature change is not conducive to the extraction of fruiting bodies, and the occurrence of sexual forms must be humidified at low temperature. After years of drum culture, the Institute of Fungi in Sanming City, Fujian Province has only grown immature and tender zygotes. They believe that the formation of Cordyceps sinensis must be completed with the participation of some active substances from bat moth larvae.

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